We hypothesize that zebrafish TLR signaling is mechanistically divergent from mammals, and predict that understanding these differences will lead to the discovery of novel TLR pathways in higher vertebrates. A critical focus of this investigation involves the immune system itself, specifically ways in which its function can be augmented, extended, and prolonged. These studies will establish the origins of the mammalian TLR pathways and ultimately provide a direction for development of new targets of investigation and new therapies for human health. In this proposal, we intend to fulfill the following SPECIFIC AIMS: 1. Discover novel zebrafish TLR signaling pathway components a. Identify the zebrafish TLR receptors that recognize viral ligands b. Identify novel interactors of the zebrafish TLR pathways via a yeast two-hybrid screen c. Determine if the interactions identified in the zebrafish also occur in the human TLR pathways These experiments will reveal new components of the zebrafish TLR pathways and will provide new targets for investigation in both human and zebrafish studies. Furthermore, the critical domains for protein-protein interaction of zebrafish TLR proteins will be elucidated. 2. Determine the role of the zebrafish antiviral pathway components in protecting the host from infection in vivo It is essential to demonstrate the link between TLR pathways and resistance to disease. In an in vivo approach, zebrafish insertional mutations and morpholino oligonucleotides (MO) will be used to knockdown the expression of the TLR pathway proteins. The loss-of-function mutants or morphants (morpholino-injected zebrafish) will then be exposed to pathogens and examined to determine how alterations in innate immune function affect resistance to pathogen challenge. PUBLIC HEALTH RELEVANCE Infectious diseases continue to play a major role in the human condition worldwide and as a result vaccines, antibiotics, and intervention strategies are continually being developed. These methods have largely proven effective;however, due to microbial adaptation and environment- triggered emergence of new diseases, it is critical to investigate alternative methods for controlling infectious diseases. A critical focus of this investigation involves the immune system itself, specifically ways in which its function can be augmented, extended, and prolonged.